Biological oil composition, formulations comprising the oil composition, and use thereof to prevent or treat cardiovascular disease

ABSTRACT

This invention relates to a biological oil composition, preferably obtained from a copepod, most preferably the copepod  Calanus finmarchicus  and the use thereof to prevent or treat formation of atherosclerotic plaques and hence development of coronary heart disease. The composition comprises the same marine n-3 polyunsaturated fatty acids (PUFAs) generally regarded as being responsible for the anti-atherosclerotic effect of marine oils, namely EPA (C20:5n-3 eicosapentaenoic acid) and DHA (C22:6n-3 docosahexaenoic acid). However, quite unexpectedly, it has been found that the oil composition of the present invention has a remarkably higher ability to prevent formation of atherosclerotic plaques than what can be attributed to EPA and DHA alone, and moreover, unlike EPA and DHA alone it has a notable blood cholesterol lowering effect.

FIELD OF INVENTION

The present invention concerns a biological oil composition,formulations comprising the oil composition, and the use of the oilcomposition in dietary supplements, functional foods and pharmaceuticalproducts for the prevention or treatment of cardiovascular disease.

BACKGROUND OF INVENTION

In the 1970's, Bang, Dyerberg and Nielsen described the plasma lipid andlipoprotein pattern of Eskimos living on the west coast of Greenland,and compared it with that of the Danish population (H. O. Bang, J.Dyerberg and A. B. Nielsen. Plasma lipid and lipoprotein pattern inGreenlandic West-coast Eskimos. Lancet 1971;1:1143-45). Later, Dyerbergand his collaborators (J. Dyerberg, H. O. Bang and N. Hjørne. Fatty acidcomposition of the plasma lipids in Greenland Eskimos. American Journalof Clinical Nutrition 1975;28:958-66) related the differences theyfound, to the remarkably low mortality from coronary heart disease amongthe Eskimos, compared to Danes. Since the dietary fat intake was almostthe same in the two populations, they suggested that the strikingdifference in coronary heart disease could be due to the big differencein the intake of marine fats and that coronary heart disease could beassociated with the chemical nature of the dietary lipids (J. Dyerberg,H. O. Bang, E. Storffersen, S. Moncada and J. R. Vane. Eicosapentaenoicacid and prevention of thrombosis and atherosclerosis? Lancet1978;2:117-19). After these pioneering studies, it became evident thatcoronary heart disease, which is still among the most serious killerdiseases in Western societies, could no longer be regarded merely as alipid storage disease caused by excessive dietary fat intake.

The scientists who pioneered this research were the first to suggestthat the anti-atherogenic factors in the traditional Eskimo diet weremarine long chain poly unsaturated fatty acids (PUFAs). Their diet,consisting largely of seal, whale and seabirds, and, to some extent,fish, provided several grams—may be as much as 15 grams—of such fattyacids each day. This is far more than a typical “modern” Western dietcontains.

Research during the last 30-40 years has confirmed the classical studiesby the Dyerberg group and established a firm scientific foundation for acommon understanding among scientists and other professionals: Thehealth benefits of sea-food and marine oils can first and foremost beassociated with two typical marine PUFAs, namely eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA). This statement is in line with theconclusions and recommendations from the symposium “Beyond Cholesterol:Prevention and Treatment of Coronary Heart Disease with n-3 Fatty Acids,published by Deckelbaum et al. (American Journal of Clinical Nutrition2008;87(suppl): 2010S-2S).

EPA and DHA contain, respectively, 20 and 22 carbon atoms with 5 and 6conjugated double bonds, of which the first one is in the position 3carbon atoms (n-3) counted from the hydrophobic (methyl) end of boththese fatty acids. The abbreviation C20:5n-3 is often used as a chemicaldesignation for EPA and C22:6n-3 for DHA. Phytoplankton in the marineenvironment is the primary producers of EPA and DHA, which follow thefood-web from this first trophic level via zooplankton to fish andsea-mammals. Plant food oils and animal fat contain low levels, if any,of EPA and DHA.

EPA and DHA are believed to be particularly important in prevention ofcardiovascular disease. Even modest sea-food intake, supplying 250 mg ofEPA and DHA daily, seems sufficient to reduce the risk of coronary deathby 36% and to reduce mortality in the general population by 17% (U. J.Jung et al. American Journal of Clinical Nutrition 2008;87(suppl):2003S-9S).

Physiological and molecular mechanisms proposed to explain thecardioprotective effects of EPA and DHA, include 1) lowering the levelsof triacylglycerol and free fatty acids in plasma, 2) increasing highdensity lipoprotein (HDL) levels and decreasing low density lipoproteins(LDL) levels, 3) decreasing platelet aggregation, 4) decreasingcholesterol delivery and cholesterol deposition in arterial walls, 5)decreasing arterial inflammation. These are interactive mechanismsinvolving complex and diverse biochemical mechanisms, including effectsof EPA and DHA as well as of their transformation products(prostaglandins, prostacycline, thromboxans, leukotrienes) on modulationof immunity and inflammation and gene expression in different cells andtissues. Although the health benefits of EPA and DHA no longer can bequestioned, the mechanisms involved are too complex to be fullyunderstood. For instance, it is still a puzzling fact that “the majormechanisms underlying the beneficial effects of n-3 fatty acids in theprevention and treatment of coronary artery disease appears to bedistinct from effects on lowering plasma triacylglycerol concentrations”(Deckelbaum et al., American Journal of Clinical Nutrition2008;87(suppl): 2010S-2S).

Preclinical and human clinical studies during the last 30-40 years haveprovided consistent evidence that consumption of sea-food and marinefood oils is beneficial for the health, and it has become generallyaccepted among those skilled in the art that these health benefits are,first and foremost, associated with EPA (eicosapentaenoic acid) and DHA(docosahexaenoic acid). The total evidence related to the ability ofthese two marine n-3 PUFAs to prevent coronary heart disease is sooverwhelming that it has become part of a primary prevention strategy ofhealth authorities in Western societies to recommend daily EPA and DHAconsumption. In support of this strategy, it can be referred to thesymposium “Beyond Cholesterol: Prevention and Treatment of CoronaryHeart Disease with n-3 Fatty Acids” summarized and discussed by R. J.Deckelbaum et al. (American Journal of Clinical Nutrition, 2008; 87(suppl): 2010S-2S). Moreover, concentrates of EPA and DHA, produced asdisclosed in U.S. Pat. Nos. 5,502,077, 5,656,667 and 5,698,594, havebeen approved by the US Food and Drug Administration (FDA) aspharmaceutical preparations that reduce the level of blood componentsregarded as risk factors for coronary heart disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representative visual illustration of plaque formation inaortas of female mice fed a diet to which was added the biological oilcomposition (Diet 1) or the EPA/DHA-concentrate (Diet 2), compared tothe reference diet (Diet 3).

FIG. 2 illustrates the average growth of female mice (n=10) on the threeexperimental diets.

FIG. 3 shows the average weight of different organs of female mice(n=10) fed the three experimental diets (WAT=white adipose tissue).

DETAILED DESCRIPTION OF THE INVENTION

In the description of the present invention below the terms biologicaloil composition, copepod oil, copepod oil composition, oil compositionare used interchangeable.

EPA and DHA are predominant fatty acids present in marine fish, whale,seal and crustaceans. Also, the oil present in the marine copepodCalanus finmarchicus is a rich source of EPA and DHA, but this oildiffers from other marine oils in a number of other chemicalcharacteristics. Compared to other marine oils, the copepod oil of thepresent invention is very rich in the C18:4n-3 PUFA (stearidonic acid,SDA). Unlike other common marine food oils, the PUFAs present in thecopepod oil exist predominantly as monoesters with long chainmonounsaturated alcohols i.e. wax esters. Compared to other commondietary marine oils, the copepod oil of the present invention contains arelatively high proportion of free fatty acids, low amounts oftriglycerides, and high levels of astaxanthin and cholesterol.

Based on the common understanding that EPA and DHA are the key factorsresponsible for the beneficial effects of marine oils in prevention andtreatment of coronary artery disease, the biomedical effects of thesetwo fatty acids have been compared with those of the copepod oilcomposition, as described in the present invention. The effects of thecopepod oil according to the present invention have been compared withthat of a concentrated EPA/DHA-preparation on atherosclerotic plaqueformation and on total cholesterol level in mice, adjusted so thatcopepod oil provided the same total amount of EPA and DHA as the totalamount of EPA and DHA in the reference preparation. In these studies,experimental animals (Apolipoprotein E (ApoE) deficient mice) feeding onan atherogenic high fat (21% w/w) diet containing 0.2% (w/w) cholesterolwere used.

Although there are differences in chemical composition between thecopepod oil composition of the present invention and other dietarymarine oils, the remarkable difference in biological activities, asdescribed in the present invention, could not at all be predicted byanyone working on the effects of marine PUFAs on coronary heart disease.Most striking is the highly unexpected finding that the biological oilcomposition of the present invention, as opposed to concentratedEPA/DHA, has a statistically significant ability to inhibit formation ofatherosclerotic plaques. It also differs from EPA and DHA in the way itaffects the pattern of lipid deposition in the body of the experimentalanimals. The copepod oil described in the present invention is in itselfa novel anti-atherosclerotic composition.

The biological oil composition according to the present invention alsoshows a significant effect on blood cholesterol level. Total bloodcholesterol levels are significantly lower in animals fed with a dietcomprising the biological oil composition according to the presentinvention as compared with the levels in animals fed a diet comprisingconcentrated EPA/DHA

The biological oil composition according to the present invention isderived from a marine copepod, preferably a copepod of the genusCalanus, such as Calanus finmarchicus, using freshly harvested,frozen/thawed or dehydrated raw material. Oil compositions according tothe invention may be obtained by any method known to the person skilledin the art such as, but not limited to, conventional fish oil productiontechnology, biotechnological methods, organic solvents or supercriticalfluid extraction, or cold pressing. Independent of the procedure ofobtaining the oil and the yield of oil, the typical gross compositionwill be as shown in Table 1. To illustrate the uniqueness of thebiological oil composition according to the present invention, thecorresponding compositions of conventional fish oil (cod liver oil) andkrill oil are shown for comparison. It is evident from this grosschemical analysis that these oils are highly different, in particularregarding their contents of triglycerides, phospholipids, monoesters(wax esters), and of astaxanthin. It should be noted that wax estersconstitute the major lipid component in the copepod oil of the presentinvention, unlike both cod liver oil and krill oil.

TABLE 1 Typical chemical composition of three different marine oils: (A)Copepod oil from Calanus finmarchicus caught in Norwegian waters, (B)cod liver oil from Atlantic cod Gadus morhua, and (C) krill oil fromEuphausia superba caught in the Southern ocean, given in mg/g oil. Lipidclasses A¹ B² C³ Triglycerides 60 955 260 Free fatty acids 80 14 13Fatty alcohols 62 0 0 Saturated fatty acids 190 160 300 Monounsaturated125 385 300 Polyunsaturated 270 475 387 n-3 fatty acids >250 395 332 n-6fatty acids <15 50 55 Cholesterol 40 12 50 Wax esters (fattyacid/alcohol esters) 650 0 0 Polar lipids (phospholipids, free 200-26018 670 fatty acids, free fatty alcohols) Neutral lipids (triglycerides,wax 740-800 967 310 esters, cholesterol) ¹Copepod oil produced byCalanus AS (www.calanus.no). ²From Falch, E., Rustad, T., and Aursand,M. By-products from gadiform species as raw material for production ofmarine lipids as ingredients in food or feed. Process Biochemistry 2006;41:666-674. ³From Phleger, C. F., Nelson, M. N., Mooney, B. D., andNichols, P. D. Interannual and between species comparison of the lipids,fatty acids, and sterols of Antarctic krill from the US AMLR ElephantIsland survey area. Comparative Biochemistry and Physiology Part B 2002;131:733-747.

Besides the notable difference in gross chemical composition (Table 1),the three marine oils used here for illustration purposes, are highlydifferent also in their content of individual fatty acids (Table 2).

TABLE 2 Fatty acid composition of three different marine oils: (A)Copepod oil from Calanus finmarchicus caught in Norwegian waters, (B)cod liver oil from Atlantic cod Gadus morhua, and (C) krill oil fromEuphausia superba caught in the Weddell Sea, given in mg/g oil. Fattyacids A¹ B² C³ 14:0 FA (myristic) 108 40 119 15:0 FA 6 0 0 16:0 FA(palmitic) 72 112 209 16:1 n-9 1.8 0 0 16:1 n-7 FA 16 61 0 16:1 n-5 FA 00 56 17:0 FA 1.7 0 0 16:2 n-4 FA 1.7 0 0 18:0 FA 4.5 27 15 16:3 n-3 0.80 0 18:1 n-9 FA (oleic) 23.4 167 170 16:4 n-3 2.2 0 0 18:1 n-7 FA 2.8 4070 18:2 n-6 FA 10.2 19 25 18:3 n-3 FA 24.4 14 9 20:0 FA 0 0 0 18:4 n-3FA (stearidonic, SDA) 109.7 21 51 20:1 n-11 FA 5.3 0 0 20:1 n-9 FA(gadoleic) 27 98 13 20:4 n-6 FA 2.0 8 7 20:4 n-3 FA 9.0 0 0 22:1 n-11(+20:4 n-3 FA) 42.7 8.5 0 22:1 n-9 FA 2.7 0 0 20:5 n-3 FA(eicosapentaenoic, EPA) 67.0 72 128 22:4 n-6 FA 10.5 0 0 24:1 n-9 FA 2.90 0 22:5 n-3 FA 3.7 20 0 22:6 n-3 FA (docosahexaenoic, DHA) 54.7 188 101Sum identified 612.7 895.5 973 ¹Copepod oil produced by Calanus AS(www.calanus.no). ²From Standal, I. B., Praël, A., McEvoy, L., Axelson,D. E., and Aursand, M. Discrimination of Cod Liver Oil According toWild/Farmed and Geographical Origins by GC and 13C NMR. J. Am Oil ChemSoc 2008; 85:105-112. ³From Hagen, W., Kattner, G., Terbrüggen, A., andVan Vleet, E. S. Lipid metabolism of the Antarctic krill Euphausiasuperba and its ecological implications. Marine Biology 2001;139:95-104.

The most noteworthy difference in fatty acid composition between thethree oils, is the very high stearidonic acid (SDA) content in thecopepod oil.

In the oil composition of the present invention, SDA, EPA and DHA existto a large extent as esters with long chain alcohols. A typicalcomposition of wax esters and long chain alcohols in the copepod oil ofthe present invention is shown in Table 3.

TABLE 3 Typical composition of wax esters and alcohol/fatty acidcombinations (% (w/w)) in copepod oil derived from Calanus finmarchicus.¹ Major alcohol/fatty Minor alcohol/fatty Wax ester acid acid % (w/w)30:1 14:0/16:1 16:1/14:0 0.8 32:1 16:0/16:1 14:0/18:1 1.9 32:2 16:1/16:114:0/18:2 0.6 32:4 14:0/18:4 16:0/16:4 0.9 34:1 16:0/18:1 14:0/20:1 —20:1/14:0 17.6 34:2 16:0/18:2 16:1/18:1 0.9 34:3 16:0/18:3 16:1/18:2 —34:4 16:0/18:4 16:1/18:3 2.7 34:5 14:0/20:5 16:1/18:4 0.4 36:1 20:1/16:016:0/20:1 — 22:1/14:0 21.9 36:2 20:1/16:1 16:1/20:1 2.3 36:5 16:0/20:520:1/16:4 1.1 36:6 16:1/20:5  14:/22:6 0.3 38:1 22:1/16:0 16:0/22:1 2.838:2 22:1/16:1 20:1/18:1 3.9 38:3 20:1/18:2 22:1/16:2 0.4 38:4 20:1/18:322:1/16:3 0.9 38:5 20:1/18:4 22:1/16:4 5.4 38:6 16:0/22:6 16:1/22:5 —40:2 20:1/20:1 22:1/18:1 5.9 40:3 22:1/18:2 0.7 40:5 22:1/18:4 20:1/20:44.7 40:6 20:1/20:5 1.5 42:2 22:1/20:1 20:1/22:1 12.7 42.6 22:1/20:520:1/22:5 1.5 42:7 20:1/22:6 2.0 44:2 22:1/22:1 4.9 44:7 22:1/22:6 0.6 ¹Compiled from Graeve, M. and Kattner, G. Species-specific differences inintact wax esters of Calanus hyperboreus and C. finmarchicus from FramStrait-Greenland Sea. Marine Chemistry 1992; 39:269-281.

In conclusion, the copepod oil of the present invention differs markedlyfrom typical fish oil and krill oil in both gross chemical compositionand fatty acid content. However, like other marine oils it comprises EPAand DHA.

In spite of its high wax-ester content, the oil composition of thepresent invention is a low-viscous and completely free-flowing liquid atroom-temperature. One of the reasons for this is that the alcohols ofthe wax esters are predominated by medium-length monounsaturatedalcohols, typically 80% or more (mainly C20:1 and C22:1).

Depending on the analytical methods used, the typical content ofwax-ester of the oil composition of the present invention is 70-90%,whereas it contains 10-20% of other components such as free fatty acids,triacylglycerols, sterols and pigments. In certain applications, it maybe advantageous or even desirable to remove free fatty acids and othercomponents by suitable methods known to those skilled in the art. Thus,in one embodiment of the preset invention the oil composition maycontain up to 100% wax ester.

It has been found that the copepod oil according to the presentinvention has markedly different biological effects than a concentratedpreparation of EPA and DHA used in the same concentration as in thecopepod oil. Particularly the composition according to the presentinvention prevents the formation of atherosclerotic plaque and thus isuseful in the prevention and treatment of cardiovascular disease. Thecomposition according to the present invention is also found to have aneffect on the total blood cholesterol level and is useful in theprevention and treatment of hypercholesterolaemia and elevated bloodcholesterol levels.

The biological oil composition according to the present inventioncomprises from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, byweight up to 75%, 80%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%,95%,96%, 97%, 98%, 99%, 100% by weight of wax esters. Preferably thebiological oil composition comprises from 80%, 81%, 82%, 83%, 84%, 85%,86%, 87% 88%, 89% by weight up to 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%,98%, 99%, 99.5%, 100% by weight of wax esters.

Further the biological oil composition of the present inventioncomprises from 5%, 6%, 7%, 8%, 9%, 10% by weight up to 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20% by weight of SDA.

The content of EPA in the biological oil composition may be 3%,4%,5%,6%, 7%, by weight up to 8%, 9%, 10%, 11%, 12%, 13%, 14%,15% byweight. The composition may comprise 2%, 3%, 4%,5% by weight up to6%,7%, 8%, 9%, 10% by weight of DHA.

In one embodiment, the present invention provides a biological oilcomposition wherein the composition comprises 20-100% by weight of waxesters, preferably 50-100% by weight of wax esters, more preferred70-100% by weight of wax esters for use as a medicament for theprevention and treatment of cardiovascular disease. The oil compositionmay be isolated from a marine copepod, preferably one of the genusCalanus, and more preferably said copepod is of the species Calanusfinmarchicus.

In other embodiments of the invention the present invention provides abiological oil composition for the use as a medicament in the preventionand treatment of atherosclerosis, hypercholesterolaemia and elevatedblood cholesterol levels.

In another embodiment the present invention provides an oil compositionthat further comprises 5-20% by weight of SDA.

In yet another embodiment the present invention provides an oilcomposition comprising 3-15% by weight of EPA and 2-10% by weight ofDHA.

In a further embodiment of the present invention an oil compositioncomprising 20-100% by weight of wax esters, preferably 70-100% by weightof wax esters, 5-20% by weight of SDA, 3-15% by weight of EPA and 2-10%by weight of DHA is provided.

In another embodiment of the present invention an oil compositioncomprising fatty alcohols and SDA, DHA and EPA as monoester with fattyalcohols is provided.

In a further embodiment the present invention provides an oilcomposition comprising 1000-4000 ppm of astaxanthin, mainly inesterified form.

A dietary supplement formulation comprising an oil composition asdescribed above is also provided by the present invention.

A functional food formulation comprising an oil composition as describedabove is also encompassed by the present invention.

In yet another embodiment of the present invention a pharmaceuticalformulation comprising an oil composition as described above isprovided.

The formulation according to the invention comprising an oil compositionas described above may be provided in capsules, tablets, emulsions ortonics and may comprise one or more pharmaceutically acceptable additiveselected from the group consisting of adjuvans, antioxidants,emulsifiers, surfactants and carriers.

The present invention further provides the use of an oil composition asdescribed above for the manufacturing of a product for the prevention ortreatment of a cardiovascular disease, particularly atherosclerosis,hypercholesterolaemia and elevated blood cholesterol levels.

The present invention also provides a method for the prophylaxis ortreatment of cardiovascular disease, particularly atherosclerosis,hypercholesterolaemia and elevated blood cholesterol levels wherein theindividual in need of such prophylactic or curative treatment is orallyadministered with a pharmaceutical composition comprising a biologicaloil composition wherein the composition comprises 20-100% by weight ofwax esters, preferably 50-100% by weight of wax esters, more preferred70-100% by weight of wax esters, and wherein a daily dosage level in therange of 4-100 mg/kg body weight.

In another embodiment the present invention provides a method whereinthe administered pharmaceutical composition further comprises 5-20% byweight of SDA.

In yet another embodiment the present invention provides a methodwherein the pharmaceutical composition comprising 3-15% by weight of EPAand 2-10% by weight of DHA.

In a further embodiment of the present invention a method wherein theadministered pharmaceutical composition comprises 20-100% by weight ofwax esters, preferably 70-100% by weight of wax esters, 5-20% by weightof SDA, 3-15% by weight of EPA and 2-10% by weight of DHA is provided.

In another embodiment of the present invention a method wherein theadministered pharmaceutical composition comprising fatty alcohols andSDA, DHA and EPA as monoester with fatty alcohols is provided.

In a further embodiment the present invention provides a method whereinthe administered pharmaceutical composition comprises 1000-4000 ppm ofastaxanthin, mainly in esterified form. The following non-limitingexperimental part and examples illustrate and document the presentinvention.

EXAMPLES

Experimental

When studying the preventive efficacy of any drug candidate or dietaryingredient on coronary heart disease, the most reliable end-pointanalyses are the actual disease manifestations, such as, for instance,formation of atherosclerotic plaques. Effects on blood parametersconsidered to be indicative of the risk of disease development are ofcourse important for evaluation of mode of action of newanti-atherogenic drug candidates, but it is preferable to relate suchblood analyses to efficacy data on the disease manifestation itself Thishas been the philosophy in the studies constituting the foundation ofthe present invention.

The biological effects of the copepod oil of this invention wererecorded in mice deficient in apolipoprotein E (ApoE). Mice of thisstrain are routinely used to determine effects of dietary components ondevelopment of vascular inflammation and atherosclerotic plaques, sincethey develop atherosclerotic lesions according to a pattern very similarto that of humans, and they are useful model animals for studies ofbiochemical and cellular processes involved in initiation, progressionand regression of atherotrombotic disease. The studies were carried outat the Faculty of Medicine at the University of Tromsø (Norway). Threegroups of ten female mice were installed at an age of 7 weeks and fed 3different diet treatments (see below) for 13 weeks.

The mice were fed ad libitum with an experimental high fat (21% w/w) andcholesterol (0.2% w/w) diet, rich in bioavailable carbohydrates(sugar/dextrin) and with a high proportion of saturated fat (sniffSpezialdiaten GmbH, sniff EF Clinton/Cybulsky (II) mod.). Thecomposition of this diet promotes development of obesity and ofatherosclerotic lesions. The diet was added either 1 (w/w) of thecopepod oil of the present invention (Diet 1) or 0.1223% (w/w) of anEPA/DHA-concentrate (Diet 2), producing two experimental feeds withequal contents of EPA and DHA. The cholesterol content of these twodiets and of the control diet (Diet 3) without added oil was adjusted to0.20% by adding cholesterol, taking into account the cholesterol presentin the feed ingredients and in the copepod-oil itself. The compositionof the experimental diets is shown in Table 4.

TABLE 4 Experimental diet for rats and mice with high fat/cholesterolcontent (type ssniff ® EF Clinton/Cybulsky (II) mod.) ¹with ingredientand nutritional profile for the three test groups Diet 1 Diet 2 Copepodoil EPA/DHA Diet 3 Ingredients preparation concentrate Control Sucrose,% 33.0876 33.0476 32.5867 Milk fat, % 19.9692 19.9692 19.9692 Casein(vitamin free), % 19.4700 19.4700 19.4700 Maltodextrin, % 9.9846 9.98469.9846 Corn starch, % 4.9923 4.9923 4.9923 Powdered cellulose, % 4.99234.9923 4.9923 AIN-76 Mineral Mix, % 3.4946 3.4946 3.4946 CalanusOil-841, % 1.0000 — — Omacor Oil-842, % — 0.1223 — AIN-76A Vitamin Mix,% 0.9985 0.9985 0.9985 Corn Oil, % 0.9985 0.9985 1.9985 Calciumcarbonate, % 0.3994 0.3994 0.3994 DL-Methionine, % 0.2995 0.2995 0.2995Choline bitartrate, % 0.1997 0.1997 0.1997 Cholesterol, % 0.1498 0.14980.1498 Ethoxyquin, % 0.0040 0.0040 0.0040 Nutritional profile Protein, %17.4 17.4 17.4 Fat, % 21.0 21.0 21.0 Cholesterol, ppm 2 027 2 027 2 027Carbohydrates, % 48.9 48.9 48.4 Fiber (max), % 5.0 5.0 5.0 Energy,kcal/g 4.48 4.56 4.55 From Protein, % 15.3 15.3 15.4 Fat (etherextract), % 41.6 41.7 41.8 Carbohydrates, % 43.0 43.0 42.8 ¹ Produced byssniff Spezialdiäten GmbH (www.ssniff.de).

The copepod oil preparation was an experimental product provided byCalanus AS, Tromsø, Norway (www.calanus.no). The EPA/DHA concentrateused as reference test substance was the lipid lowering drug Omacor(Pronova Biopharma ASA, P.O. Box 420, NO-1327 Lysaker, Norway).According to the manufacturer (www.pronova.com) this product contains90% omega-3-acid ethyl esters of EPA (460 mg/g) and DHA (380 mg/g) andis manufactured using fish oil as a starting material.

The experimental mice were monitored daily, and weighed at regularintervals. Samples of blood serum were taken at different points forlater analysis of various blood parameters including lipids and fattyacids. The mice were sacrificed at the end of the experiment, and allrelevant organs were dissected out following standard procedures.Following dissection of the sacrificed mice, the aortas were isolated,cleaned and cut open longitudinally, pinned to a white cardboard andfixed in 10% formalin for at least 24 hours. The aortas were stainedwith Oil Red O (Sigma) before analysis. After rinsing, the aortas weremounted on microscopic slides, and images (2,700 dpi) were acquiredusing a SprintScan 35 scanner (Polaroid, Cambridge, Mass., USA) equippedwith GeoScan Enabler (Meyer Instruments, Houston, Tex., USA). The imageswere analyzed for positive areas, adopting the state-of-the artcalibration and image analyses methodology. The total lesion area wasquantified in each group by computer-assisted quantitative morphometryas described by N. V. Guevara et al. (The absence of p53 acceleratesatherosclerosis by increasing cell proliferation in vivo. NatureMedicine 1999; 5:335-339).

Biological Effects i) Atherosclerosis

It has been found that the copepod oil of the present invention hasmarkedly different biological effects than a concentrated preparation ofEPA and DHA used in the same concentration as in the copepod oil. Thiswas a highly unexpected finding, considering the overwhelming consensusamong the skilled in the art, that the positive health effects of marineoils are associated with their content of EPA and DHA, exclusively.

The results are shown in Table 5 and in FIGS. 1-3.

The effect of the copepod oil of the present invention and ofEPA/DHA-concentrate on atherosclerotic plaque formation in the aortas offemale mice is shown in Table 5. The copepod oil preparation had astriking and statistically highly significant effect on reduction ofplaque formation both in the aortic arch (p=0.002) and the total aorta(p=0.001) compared to control. Also the EPA/DHA-concentrate reducedplaque formation compared to control, but the effect did not meet therequirements of statistical significance.

TABLE 5 The effect of a copepod oil preparation and of concentratedEPA/DHA on atherosclerotic plaque formation¹ in the ascending aorticarch, thoracal, abdominal and perirenal segments of the aorta in femalemice. Diet 1 Diet 2 Copepod oil EPA/DHA Diet 3 preparation concentrateControl Target region (n = 10) (n = 10) (n = 10) Aortic arch (A) 15.118.0 22.0 Thoracal (B) 7.93 9.51 12.16 Abdominal (C) 1.93 2.52 3.94Perirenal (D) 1.36 2.27 1.94 Total aorta (B-D) 4.59 5.87 7.22 ¹Thefigures represent the average lesion area in percent of total area ofeach target region at time of sacrifice. See FIG. 1 for the subdivisionof target regions (A-D) of the aorta.

Growth of the mice is shown in FIG. 2. Although the mice grew fastest onfeed enriched with copepod oil, and thrived well on that diet, thisapparent difference does not meet the requirements for statisticalsignificance. There was no difference between the groups in feed intakeand no negative effects could be observed on animals fed theexperimental diets.

Weight of different organs is shown in FIG. 3. Although there was ahigher level of fat deposited in the white adipose tissue (WAT) in micefed the copepod oil, the difference was not statistically significant.However, it is a noteworthy observation indeed that the copepod oil ofthe present invention reduces plaque formation while more lipids aredeposited in lipid storage tissues.

ii) Blood Cholesterol Level

The copepod oil has a notably more pronounced anti-atheroscleroticeffect than purified EPA and DHA at same concentration as in this oil.The mechanisms involved in this effect of the copepod oil mayaccordingly be additive to the EPA- and DHA-effects or be entirelydifferent.

The results shown in Table 6 illustrate that Calanus Oil differs fromEPA and DHA also regarding the effect on blood cholesterol level in theexperimental animals.

Whereas the cholesterol level in blood of animals fed the EPA/DHA-dietwas the same as in control animals after 13 weeks of feeding, thecholesterol level in blood of the Calanus Oil group was notably lower.Both treatment groups seem to have a slight, and similar, triglyceridelowering effect compared to control.

TABLE 6 Effects of the dietary supplements on bodyweight, food intakeand plasma lipids in apoE-deficient female mice after 13 weeks oftreatment, as mean values +− SEM. Diet 1 Diet 2 Copepod oil EPA/DHA Diet3 Female apoE- composition concentrate Control deficient mice (n = 10)(n = 10) (n = 10) Bodyweight (g) Initial 18.4 +/− 0.3  18.7 +/− 0.3 18.6 +/− 0.4  Final 38.9 +/− 1.2  37.7 +/− 1.6  34.6 +/− 1.2  Foodintake 2.72 +/− 0.05 2.72 +/− 0.07 2.77 +/− 0.04 (g/day) Totalcholesterol 12.3 +/− 1.25 15.9 +/− 1.28 16.1 +/− 1.25 (mmol/L)Triacylglycerol 0.82 +/− 0.05 0.84 +/− 0.07 0.96 +/− 0.05 (mmol/L)

1. A biological oil composition comprising 20-100% by weight of waxesters, preferably 70-100% by weight of wax esters for use as amedicament for the prevention or treatment of a cardiovascular disease.2. The biological oil composition according to claim 1 for use as amedicament for the prevention or treatment of atherosclerosis.
 3. Thebiological composition according to claim 1 for use as a medicament forthe prevention or treatment of hypercholesterolaemia.
 4. The biologicalcomposition according to claim 1 for the use as medicament for theprevention or treatment of elevated blood cholesterol levels.
 5. Thebiological oil composition according to claims 1 to 4 wherein thecomposition comprises 5-20% by weight of stearidonic acid (SDA).
 6. Thebiological oil composition according to any one of claims 1 to 5 whereinthe composition comprises 3-15% by weight of eicosapentaenoic acid (EPA)and 2-10% by weight of docosahexaenoic acid (DHA).
 7. The biological oilcomposition according to any one of claims 1 to 6 wherein thecomposition comprises fatty alcohols and SDA, DHA and EPA as monoesterswith fatty alcohols.
 8. The biological oil composition according to anyone of claims 1 to 7 wherein the composition contains 1000-4000 ppm ofastaxanthin.
 9. The biological oil composition according to any one ofclaims 1 to 8 wherein said composition is isolated from a copepod. 10.The biological oil composition according to claim 9 wherein said copepodis of the genus Calanus.
 11. The biological oil composition according toclaim 10 wherein said copepod is of the species Calanus finmarchicus.12. Dietary supplement formulation comprising a biological oilcomposition according to any one of claims 1-11.
 13. Functional foodformulation comprising a biological oil composition according to any oneof claims 1-11.
 14. A pharmaceutical formulation comprising a biologicaloil composition according to any one of claims 1-11.
 15. A formulationaccording to claim 12 or 14 provided in capsules, tablets, emulsions ortonics.
 16. A formulation according to claim 15 comprising one or morepharmaceutically acceptable additives selected from the group consistingof adjuvans, antioxidants, emulsifiers, surfactants and carriers.